1. Field of the Invention
This invention is in the field of human medicine. More particularly, this invention is in the field of pharmaceutical treatment of the diseases of diabetes and hyperglycemia.
2. Description of Related Art
It has long been a goal of insulin therapy to mimic the pattern of endogenous insulin secretion in normal individuals. The daily physiological demand for insulin fluctuates and can be separated into two phases: (a) the absorptive phase requiring a pulse of insulin to dispose of the meal-related blood glucose surge, and (b) the post-absorptive phase requiring a sustained delivery of insulin to regulate hepatic glucose output for maintaining optimal fasting blood glucose.
Accordingly, effective therapy for people with diabetes generally involves the combined use of two types of exogenous insulin formulations: a rapid acting meal time insulin provided by bolus injections and a long-acting, so-called, basal insulin, administered by injection once or twice daily to control blood glucose levels between meals. An ideal basal insulin will provide an extended and xe2x80x9cflatxe2x80x9d time actionxe2x80x94that is, it will control blood glucose levels for at least 12 hours, and preferably for 24 hours or more, without significant risk of hypoglycemia. Furthermore, an ideal basal insulin should be mixable with a soluble meal-time insulin, and should not cause irritation or reaction at the site of administration. Finally, basal insulin preparations that are suspension formulations should be able to be readily, and uniformly resuspended by the patient prior to administration.
As is well understood by those skilled in this art, long-acting insulin formulations have been obtained by formulating normal insulin as microcrystalline suspensions for subcutaneous injection. Examples of commercial basal insulin preparations include NPH (Neutral Protamine Hagedorn) insulin, protamine zinc insulin (PZI), and ultralente (UL).
Early versions of present-day commercial NPH insulin that contained a surplus of protamine were developed in the 1930""s by Scott, et al. [J. Pharmacol. Exp. Ther. 58:78, et seq. (1936)] and Hagedorn, et al. [J. Am. Med. Assoc. 106:177-180 (1936)]. In 1946, NPH insulin having isophane proportions of insulin and protamine, together with zinc, were developed by Krayenbuhl, et al. [Rep. Steno Mem. Hosp. Nord. Insulinlab. 1:60, et seq. (1946)]. These workers found that when insulin and protamine were combined in so-called isophane proportions at a neutral pH, in the presence of zinc and a phenolic compound, that an amorphous precipitate formed, and that upon standing the amorphous precipitate was transformed into oblong, tetragonal crystals having pyramidal shaped ends. These crystals have been described as rod-like. The isophane ratio of insulin and protamine sulfate is observed to be about 0.09 mg of protamine sulfate per mg of insulin. Zinc is needed in an amount of at least about 3.5 xcexcg per mg of insulin, and a phenolic compound at a concentration higher than about 0.1%.
Insulin NPH is the most widely-used insulin preparation, constituting from 50 to 70 per cent of the insulin used worldwide. It is a suspension of a microcrystalline complex of insulin, zinc, protamine, and one or more phenolic preservatives. NPH insulin preparations are commercially available incorporating human insulin, pork insulin, beef insulin, or mixtures thereof. Also, NPH-like preparations of a monomeric insulin analog, LysB298, ProB29-human insulin analog, are known in the art [abbreviated herein as xe2x80x9cNPLxe2x80x9d: De Felippis, M. R., U.S. Pat. No. 5,461,031, issued Oct. 24, 1995; De Felippis, M. R., U.S. Pat. No. 5,650,486, issued Jul. 22, 1997; and De Felippis, M. R., U.S. Pat. No. 5,747,642, issued May 5, 1998]. It is widely accepted that insulin NPH provides extended control of blood glucose compared with regular insulin because insulin must first dissolve from the insulin NPH microcrystals before it can be absorbed. With regular insulin, there is no dissolution needed prior to absorption. For insulin NPH, dissolution is the rate-controlling step in determining the pharmacodynamics and pharmacokinetics.
NPH insulin microcrystals possess a distinctive rod-shaped morphology of typical dimensions about 5 microns long by 1 micron thick and 1 micron wide. The extended duration of action of NPH insulin microcrystals results from their slow absorption from the subcutaneous injection site.
Therapy using currently-available NPH insulin preparations fails to provide the ideal xe2x80x9cflatxe2x80x9d pharmacokinetics necessary to maintain optimal fasting blood glucose for an extended period of time between meals. Consequently, treatment with NPH insulin can result in undesirably high levels of insulin in the blood, which may cause life-threatening hypoglycemia.
In addition to failing to provide an ideal flat pharmacokinetic profile, the duration of action of NPH insulin also is not ideal. In particular, a major problem with NPH therapy is the xe2x80x9cdawn phenomenonxe2x80x9d which is hyperglycemia that results from the loss of effective glucose control overnight while the patient is sleeping. These deficiencies in glycemic control contribute to serious long-term medical complications of diabetes and impose considerable inconvenience and quality-of-life disadvantages to the patient.
Protamine zinc insulin (PZI) has a composition similar to NPH, but contains higher levels of protamine and zinc than NPH. PZI preparations may be made as intermediate-acting amorphous precipitates or long-acting crystalline material. PZI, however, is not an ideal basal insulin pharmaceutical because it is not mixable with a soluble meal-time insulin, and the high zinc and protamine can cause irritation or reaction at the site of administration.
Human insulin ultralente is a microcrystalline preparation of insulin having higher levels of zinc than NPH, and not having either protamine or a phenolic preservative incorporated into the microcrystal. Human ultralente preparations provide moderate time action that is not suitably flat, and they do not form stable mixtures with insulin. Furthermore, they are difficult to resuspend.
There have been attempts to address the perceived inadequacies of known insulin suspensions. Fatty acid-acylated insulins have been investigated for basal control of blood glucose [Havelund, S., et al., WIPO publication WO95/07931, Mar. 23, 1995]. Their extended time action is caused by binding of the fatty acyl portion of these molecules to serum albumin. The fatty acyl chain lengths of these molecules is such as to take advantage of the fatty acid binding capability of serum albumin. The fatty acid chains used in fatty acid-acylated insulins are typically longer than about ten carbon atoms, and chain lengths of fourteen and sixteen carbon atoms are optimal for binding to serum albumin and extending time action.
Unlike NPH insulin, which is insoluble, the aforementioned fatty acid-acylated insulins are soluble at the usual therapeutic concentrations of insulin. However, the time action of these preparations may not be sufficiently long enough, or flat enough, to provide ideal basal control, and they are less potent than insulin, thereby requiring administration of greater amounts of the drug agent [Radziuk, J., et al., Diabetologia 41:116-120, 489-490 (1998)].
Whittingham, J. L., et al. [Biochemistry 36:2826-2831 (1997)] crystallized B29-Nxcex5-tetradecanoyl-des(B30)-human insulin analog as a hexamer complex with zinc and phenol for the purpose of structural studies by X-ray crystallography. The hexamer was found to be in the R6 conformation, and to have certain properties different from hexamers of human insulin. Whittingham, et al. do not disclose any pharmaceutical or pharmacological properties of the crystal that was formed, nor do they suggest that such a crystal would have any advantageous properties for treating diabetes or hyperglycemia. It is not possible to predict from Whittingham, et al. whether protamine-containing crystals of the NPH type could be formed with derivatized insulins and insulin analogs, or what the pharmacokinetics or pharmacodynamic response of such crystals would be.
Thus, there remains a need to identify insulin preparations that have flatter and longer time action than NPH insulin, that are mixable with soluble, meal-time insulins, that can be readily resuspended, and that do not pose risk of irritation or reaction at the site of administration. I discovered quite surprisingly that these properties are provided by insoluble compositions that include a derivatized protein, an un-derivatized protein, zinc, protamine, and a phenolic preservative. In addition to the properties mentioned above, the insoluble compositions provide flexibility of control over the duration and shape of the glucodynamic response profile. They are thought to function as controlled release compositions, wherein, the release rate is controlled by the proportion and nature of the derivatized protein. Thus, one aspect of the present invention is an insoluble composition comprising an un-derivatized protein, a derivatized protein, a complexing compound, a hexamer-stabilizing compound, and a divalent metal cation. Other aspects of this invention that relate to the preparation, formulation, and use of such compositions will be discussed herein.
There are no examples known to me of mixtures of derivatized and un-derivatized insulins, as those terms are to be understood in the context of the present disclosure. Crystals comprised of proinsulin and insulin [Steiner, D. F., Nature 243:528-530 (1973); Low, B. W., et al., Nature 248:339-340 (1974)] and crystals comprised of a insulin or an insulin analog having approximately the same isoelectric point as insulin and an insulin analog having additional basic amino acids [Dorschug, M., et al., U.S. Pat. No. 5,028,587, issued Jul. 2, 1991] are known.
Steiner produced crystals comprised of proinsulin and insulin with mole ratios of about 1:11, 1:5, 1:2, and 1:1, respectively (i.e., 0.5, 1, 2, and 3 moles of proinsulin per 6 moles total insulin and proinsulin) in 0.095 M sodium citrate, pH 6.0, 0.03 M NaCl, 0.012 M ZnCl2, and 16% acetone., The proportion of proinsulin greatly affected the rate of crystallization. The crystals differed greatly from those of pure insulin under the same conditions, and were characterized as rhombohedral crystals with rounded borders. There was great variability within and between preparations. The utility ascribed to crystallizing proinsulin and insulin was that it facilitated isolating small amounts of proinsulin and related structures from pancreatic extracts. The author speculated that crystallization may occur between precursor and product peptides, and among other closely related proteins.
Low, B. W., et al. produced very large crystals comprised of equimolar proportions of beef or pork insulin and their respective proinsulins, wherein the proinsulin and insulin were formed into homogenous hexamers prior to crystallization. Analysis by X-ray crystallography and quantitative electrophoresis supported a conclusion that the unit cell in the crystals was formed of twelve insulin hexamers and twelve proinsulin hexamers. It was specifically stated that no studies were known to suggest that insulin and proinsulin form mixed dimers and hexamers in solution.
Dxc3x6rschug, M., et al. disclosed crystals comprised of insulin, des(PheB1) insulin, des(ThrB30) human insulin, or des(AlaB30) beef insulin, and at least one insulin having a basic modification at the C-terminal end of the B chain (xe2x80x9cmodified insulinxe2x80x9d). Such modified insulins are disclosed, for example, in European Patent Application No. 132,769. Globin or protamine sulfate were stated to be auxiliary compounds that could be used in the crystal preparations. There are no examples of the use of protamine, nor any suggestion that the inventors appreciated the effect of adding such compounds. Furthermore, the modified insulins used in Dorschug, et al. are different than the derivatives used in the present invention.
As mentioned above, I have unexpectedly observed that when a protein selected from insulin, an insulin analog, and proinsulin is made less soluble in an aqueous solvent or more lipophilic by derivatizing one or more of its reactive side groups, the derivatized protein and an un-derivatized protein selected from insulin, an insulin analog, and proinsulin can be incorporated into insoluble precipitates and into NPH-like crystals with protamine. When such proteins are jointly precipitated or crystallized to form insoluble compositions, the rate at which the proteins dissolve from the insoluble composition is greatly reduced compared with the rate at which physically similar insoluble compositions comprised of un-derivatized protein dissolve.
I have furthermore discovered that both amorphous precipitates and microcrystals comprised of derivatized protein, protein, a complexing compound, a divalent metal cation, and a hexamer-stabilizing compound provide flatter and longer time action than do physically similar microcrystals comprised solely of un-derivatized protein. Additionally, I have surprisingly discovered that the benefits of flatter and longer time action can be obtained even from amorphous precipitates comprised of one of the proteins and a derivatized protein.
Accordingly, in its broadest aspect, the present invention provides insoluble compositions comprising a derivatized protein selected from the group consisting of insulin derivatives, insulin analog derivatives, and proinsulin derivatives, a protein selected from the group consisting of insulin, insulin analogs, and proinsulins, a complexing compound, a hexamer-stabilizing compound, and a divalent metal cation. The derivatized protein is either less soluble in an aqueous solvent than is the un-derivatized protein, is more lipophilic than un-derivatized insulin, or produces a complex with zinc and protamine that is less soluble than the corresponding complex with the un-derivatized protein. The insoluble compositions of the present invention may be in the form of amorphous precipitates, or more preferably, in the form of microcrystals. The microcrystals may be either rod-shaped or irregular in morphology. These insoluble compositions are useful for treating diabetes and hyperglycemia, and provide the advantages of having flatter and longer time action than NPH insulin. The insoluble compositions are mixable in a formulation with soluble protein or with soluble derivatized protein, or both. Furthermore, by varying the ratio between protein and derivatized protein, the extent of protraction of the time action can be finely controlled over a very great range of time-action, from that nearly the same as NPH insulin to much greater than that of NPH insulin.
More specifically, the present invention provides insoluble compositions of proteins and fatty acid-acylated proteins that are useful for treating diabetes and hyperglycemia. These compositions are comprised of fatty acid-acylated protein selected from the group consisting of fatty acid-acylated insulin, fatty acid-acylated insulin analog, and fatty acid-acylated proinsulin, protein selected from the group consisting of insulin, insulin analogs, and proinsulin, protamine, a phenolic preservative, and zinc. The present invention is distinct from previous fatty acid-acylated insulin technology in that the extension of time action of the present invention does not rely necessarily on albumin-binding, though albumin binding may further protract the time action of certain of the compositions of the present invention.
The invention provides a microcrystal comprising a protein selected from the group consisting of insulin, insulin analog, and proinsulin, a derivatized protein selected from the group consisting of derivatized insulin, derivatized insulin analog, and derivatized proinsulin, a complexing compound a divalent metal cation, and a hexamer-stabilizing compound. The microcrystals of the present invention are useful for treating diabetes and for controlling blood glucose in a patient in need thereof.
The invention provides an amorphous precipitate comprising a protein selected from the group consisting of insulin, insulin analog, and proinsulin; a derivatized protein selected from the group consisting of derivatized insulin, derivatized insulin analog, and derivatized proinsulin, a complexing compound a divalent metal cation, and a hexamer-stabilizing compound. The amorphous precipitates of the present invention are useful for treating diabetes and for controlling blood glucose in a patient in need thereof. They are also useful as intermediates in the formation of the microcrystals of the present invention.
The invention provides aqueous suspension formulations comprising an insoluble composition and an aqueous solvent. One such aqueous suspension formulation is comprised of a microcrystalline composition of the present invention and an aqueous solvent. Another such aqueous suspension formulation comprises an amorphous precipitate of the present invention and an aqueous solvent. The soluble, aqueous phase of the present suspension formulations may optionally be comprised of a protein, such as human insulin, or a soluble analog of human insulin, such as a monomeric insulin analog, that control blood glucose immediately following a meal, and may additionally or alternatively be comprised of a derivatized protein. The formulations of the present invention have superior pharmacodynamics compared with human insulin NPH, and their time-action can be purposefully selected over a wide range, from just slightly extended compared with human insulin NPH to very greatly extended compared with human insulin NPH.
The invention also provides processes for preparing hybrid hexamers, mixed hexamers, the amorphous precipitates, and the co-crystals of the present invention,
The invention provides a method of treating diabetes or hyperglycemia comprising, administering to a patient in need thereof a sufficient quantity of an insoluble composition of the present invention to regulate blood glucose levels in the patient.
The invention includes hybrid hexamer compositions comprising a protein selected from the group consisting of insulin, insulin analog, and proinsulin; a derivatized protein selected from the group consisting of derivatized insulin, derivatized insulin analogs, and derivatized proinsulins, and zinc. The hybrid hexamers of the present invention are useful for treating diabetes and for controlling blood glucose in a patient in need thereof. They are also useful as intermediates in the formation of the insoluble compositions of the present invention, which are themselves useful for treating diabetes and for controlling blood glucose in a patient in need thereof. Hybrid hexamers are believed to be formed when a protein and a derivatized protein are first mixed together under conditions that strongly favor dissolution into lower states of aggregation than the hexameric state, and second, the conditions are changed to strongly favor the hexameric aggregation state.
The invention includes mixed hexamer compositions, comprised zinc hexamers of a protein selected from the group consisting of insulin, an insulin analog, or proinsulin and zinc hexamers of a derivatized protein selected from the group consisting of a derivatized insulin, derivatized insulin analog, or a derivatized proinsulin. The mixed hexamers of the present invention are useful for treating diabetes and for controlling blood glucose in a patient in need thereof. They are also useful as intermediates in the formation of the insoluble compositions of the present invention, which are themselves useful for treating diabetes and for controlling blood glucose in a patient in need thereof. Mixed hexamers are believed to be formed when a protein and a derivatized protein are first separately dissolved under conditions that favor the hexameric aggregation state, and then are mixed together under conditions that continue to strongly favor the hexameric aggregation state.
The invention includes the use of an insoluble composition of the present invention to prepare a medicament for the treatment of diabetes or hyperglycemia.